Many diagnostic, research, and development procedures require the isolation and detection of specific nucleic acid (DNA or RNA) sequences present in a biological sample. For example, nucleic acid detection methods are used to identify bacteria, viruses, or other microorganisms, whose presence can indicate the cause of an infectious disease. The nucleic acids of the cells of more complex organisms, such as the DNA of human white blood cells, are also more and more commonly isolated and tested in order to establish the presence of a mutation associated with cancer or a genetic disease. Nucleic acids isolated from samples of biological tissue, such as blood taken directly from an individual or from a crime scene, are also often used to determine the identity of the individual from which the sample originated, as, for example, for paternity or forensic work. Nucleic acids are also isolated from biological tissue in order to perform research and development procedures such as cloning and nucleic acid analysis techniques well known to one skilled in the art.
Before one can isolate a nucleic acid material for any of the purposes noted above, it is necessary to make available the specific nucleic acid of interest in a sample of biological material. Frequently, the nucleic acid will be contained within a bacteria cell, a fungal cell, a viral particle, or the cell of a more complex organism, such as a human white blood cell or a plant cell.
Such cells or particles can be treated chemically or enzymatically to dissolve or denature the walls of such organisms, causing the nucleic acids to be released. This process of dissolution is commonly referred to as "lysis". The resulting solution containing such lysed material is referred to as a "lysate".
Unfortunately, such release exposes nucleic acids to degradation by endogenous nucleases present in the sample, which may exist in such abundance that destruction of the nucleic acids begins immediately upon nucleic acid release. Any nucleases remaining at the end of any subsequent purification process can continue to degrade remaining intact nucleic acids until there is nothing useful left of the original sequence of each such nucleic acid molecule. Nucleases are abundant in most biological samples and are often extremely resistant to treatments which are known to inactivate other enzymes. Deoxyribonucleases (DNases) such as endonuclease I, are naturally produced in large quantities by many of the most popular strains of bacterial cells used in cloning, transformation, and testing of DNA today. See, for example, discussion and list of end A+ strains of Escherichia coli (E. coli) by Schoenfeld et al. in Promega Notes 53: 13-21 (1995). Ribonucleases (RNases) also are abundant in most, if not all, biological samples.
Other proteins released in the process of lysing biological material can usher in another set of problems. Endotoxins, a type of lipopolysaccharide modified protein released from many types of biological material, are toxic to animal tissue culture cells, and can kill target cells before a nucleic acid-containing sequence of interest can be transformed into the cells. Thus, endotoxins can render a solution of nucleic acids contaminated therewith useless for the transfection of tissue culture cells, as the cells must be kept alive to be of any use. In addition, endotoxins also cause complications in therapy including possible introduction of nucleic acids into live animals.
To deal with the problem of nucleases and other undesirable products released by lysis, it is common in the art to employ a variety of means to purify nucleic acids from a biological sample. For example, anionic detergents and chaotropic agents, such as guanidinium thiocyanate, have been used simultaneously to inactivate or to inhibit nuclease activities and to release nucleic acids from within cells and subcellular structures. Unfortunately, many such agents are also potent inhibitors of the enzymes used in many standard procedures such as restriction digestion, transformation, amplification, targeting, and hybridization procedures. As a result of all the above factors, it has become customary to use additional isolation steps to remove these agents to recover usable, substantially intact, nucleic acids.
One common procedure used to isolate nucleic acids from a lysate is to precipitate the nucleic acids out of the solution, using a low molecular weight alcohol. Because other macromolecules also precipitate under these conditions producing a sticky, intractable mass that entraps the nucleic acids, it has frequently been necessary to resort to extraction of the sample with hazardous organic solvent mixtures containing phenol, and/or chloroform prior to ethanol precipitation. In some cases when anionic detergents are used, proteases that are active in the presence of these detergents, such as proteinase K, are used to degrade partially protein components of the sample or to degrade components that may not be extracted by the solvent treatment.
It will be readily appreciated that the method of isolation cited above is tedious, hazardous, labor-intensive, and slow. If great care is not taken in performing the procedure, residual contamination with nucleases can occur, and the sample nucleic acids will be degraded or lost. Diagnostic tests performed with such samples can also give false negative results due to such degradation. False negative results can also be obtained due to chemical interference, for example from residual anionic detergents, chaotropic salts, or ethanol remaining in the sample and inhibiting target amplification procedures. If anionic detergents and proteases have been used, residual proteolytic activity can also degrade the enzymes used in target amplification and/or hybridization detection reactions and produce false negative results. Thus, such procedures are not well suited for routine processing of biological specimens received in clinical or forensic laboratories in any quantity.
Less tedious methods of isolating nucleic acids are also known. One such method commonly used to isolate and to purify RNA uses magnetic particles, such as paramagnetic particles, to isolate specific species of nucleic acids from a lysate solution containing guanidinium thiocyanate and an anionic detergent. See, for example, PolyATtract.RTM. mRNA Isolation Systems as described in Promega Corporation's 1996 Catalog, pp. 158-160; or see PCT Publication No. WO 96/09308. Another type of nucleic acid isolation method uses silica to isolate plasmid DNA from a bacterial lysate solution containing a guanidinium salt and a base. Boom et al, J. Clinical Microbiol. 28(3): 495-503 (1990). Several silica based resins are commercially available for use in such methods. For example, a specialized silica-based resin, such as one of the Wizard.TM. DNA Purification System resins (commercially available from Promega Corporation, Madison, Wis., U.S.A.) is added to the lysate, and is allowed to bind to the nucleic acid of interest such as plasmid DNA. The resin is then loaded onto a column, washed several times using a vacuum or centrifugal force, and the nucleic acid bound to the resin is then eluted from the column with an elution buffer or water.
Although paramagnetic particle and resin methods such as those outlined immediately above are very rapid and selective methods for isolating nucleic acids, neither guarantees the inactivation of nucleases or other harmful proteins at any point during the procedure. In fact, nucleases can even be carried over into the final solution of isolated nucleic acids produced using several such methods. Nuclease carryover can result in severe degradation of nucleic acids isolated with at least the second, resin based, method of purification, particularly in cases where the DNA at issue is isolated from an end A+ bacterial strain. See, e.g. Schoenfeld et al., supra.
As is noted above, proteases have been used to enzymatically degrade proteins in nucleic acid isolation procedures. However, until now, all proteases used to isolate nucleic acids have been inactive in the alkaline pH ranges present in most alkaline lysates. For example, proteinase K is relatively inactive at pH 9 or above, and completely inactive at any pH above pH 10.5, the pH range of a typical alkaline lysate. One the other hand, proteinase K has optimal activity in the approximately neutral pH range (pH 7-8) generally used in restriction digestion or amplification reactions, where residual protease activity in a nucleic acid preparation can degrade enzymes added to the DNA.
Acid proteases, another type of protease whose use in nucleic acid isolation is known, create another host of problems. For example, U.S. Pat. No. 5,386,024 issued to Kacian et al. on Jan. 31, 1995 ("the '024 Patent"), describes a method for "using an acid protease to make available a desired nucleic acid(s) contained in a biological sample". ('024 Patent, claim 1.) The method of the '024 Patent consists of reducing the pH of a biological sample "to a pH below that at which the endogenous nucleases present in the sample are active, and adding a protease active at that low pH which degrades any nuclease that have not been irreversibly inactivated by exposure to low pH, and then inactivating the protease by raising the pH". ('024 Patent, col. 3, lines 5-11). The nucleic acid components of biological samples treated with acid proteases according to the method of the '024 Patent are available for direct use in various detection methods, without further isolation. ('024 Patent, col. 6, lines 39-41). However, Kacian also notes that the low pH used in their acid protease method can cause depurination and chain breakage in DNA ('024 Patent, col. 6, lines 4-6.) Thus, although this last method is well-suited to RNA isolation, its use to isolate intact DNA is limited.
Alkaline proteases (i.e., proteases which are active at a pH of at least 10) have been used in the detergent industry for many years to boost the washing performance of laundry and other commercial detergent formulations. See, for example, Von der Osten et al., J. Biotechnol. 28: 55-68 (1993); and Aehle et al., J. Biotechnol. 28: 31-40 (1993). Alkaline proteases purified from Bacillus licheniformis (B. licheniformis) and Bacillus alcalophilus (B. alcalophilus) are widely used in detergent formulations, being particularly favored for their low toxicity compared to proteases from other organisms, their activity at alkaline pH values, and their compatibility with detergents. Such proteases are produced cheaply and in large quantities for use in the detergent industry. For an example of one of many patented methods used to prepare and to purify alkaline proteases from these last two organisms, see U.S. Pat. No. 5,439,817 issued to Shetty et al. on Aug. 8, 1995.
The present invention addresses the problem of degradation of nucleic acids after release of the nucleic acid and protein components of biological materials during lysis in the presence of an alkaline pH. The present invention also addresses the problem of protein carryover, particularly the carryover of endotoxins and nucleases, in a variety of different nucleic acid isolation procedures. The present invention uses an alkaline protease conveniently to inactivate and degrade nucleases in biological samples, while making the nucleic acids in the sample available for further isolation using any one of a number of different known methods for nucleic acid isolation. Addition of alkaline protease to biological samples according to the method of this invention also leaves the nucleic acids in the sample sufficiently free of inhibitory or degrading enzymes to be used directly for restriction digestion, DNA sequencing, cloning and detection assays, such as hybridization assays or target amplification procedures. The present invention is a quick, simple, and relatively non-hazardous method of removing deleterious proteins from solution, and ensuring that intact and usable nucleic acids are recovered when further nucleic acid isolation is desired. The method of this invention also offers a rapid and efficient means for digesting proteins such as nucleases, which can inhibit or damage nucleic acids, limiting or even completely destroying the usefulness of the nucleic acids so isolated.
The present invention also addresses the need for methods of treating solutions of nucleic acids which contain nucleases capable of degrading the nucleic acids. This embodiment of the method of this invention offers a rapid and efficient means for inactivating the nuclease components of such solutions, thereby protecting the nucleic acids contained therein from degradation or damage by the nucleases.